ABSTRACTBackground: Low nadir CD4 cell count and apolipoprotein E4 (ApoE4) allele are known risk factors for HIV-related neurocognitive impairment. ApoE4 is also predictive of development or progression of Alzheimer’s disease (AD). The severity of dementia in HIV infection has been correlated to diffusion alterations within the corpus callosum, and in AD, with regionally specific atrophy of the same structure. We hypothesize that volumetric changes in callosal regions may correlate with nadir CD4, presence of an ApoE4 allele, and neurocognitive deficits. Methods: Seventeen HIV-seropositive men over age 50 (8 with at least one ApoE4 allele; 9 without ApoE4) underwent T1-weighted structural magnetic resonance imaging at 3.0 Tesla, clinical and neuropsychological tests, and ApoE4 genotyping. Nadir CD4 count was determined by patient self-report and confirmed as possible. We utilized FreeSurfer software to parcellate corpus callosum into five segments. Analysis of covariance was used to study the effects of callosal volumes on cognitive domains where neuropsychological test performance differed significantly (p<.05) between ApoE4-positive and non-ApoE4 groups. Independent variables were age, education, ApoE4 group status, and callosal segment volume. Regional volumes that were associated with cognitive deficit were examined for correlation with nadir CD4. Results: Education and viral load did not differ significantly between ApoE4-positive and non-ApoE4 groups. Reduction of normalized total and regional callosal volumes in ApoE4 carriers did not reach significance. Significant ApoE4-associated impairment included diminished visuospatial function and non-verbal memory, which showed effects at significant or trend (p<.1) levels of splenium (posterior), genu (anterior), and central callosal volume. Age and education did not contribute significantly to the model. Lower nadir CD4 correlated with decreased splenium (p=0.048, R=0.712) and genu (p=0.040, R=0.730) volume in ApoE4 carriers only. Conclusions: HIV-seropositive individuals possessing at least one ApoE4 allele are more vulnerable to effects of low nadir CD4 count, which may contribute to region-specific corpus callosum atrophy and thereby to cognitive impairment. Early antiretroviral therapy may be more vital for these patients than for those without the allele.

CONCLUSIONS

METHODS

• The study population:• All participants were HIV-seropositive and on highly active antiretroviral therapy (HAART) • 8 patients (ApoE4-positive group) who had at least one E4 allele were selected from a larger cohort• 9 individuals (ApoE4-negative or “control” group) did not carry the ApoE4 allele• HIV RNA, or “viral load”, was undetectable (< 50 copies/106 cells) in 15 patients, and borderline (52 copies/106 cells) or low (177 copies/106 cells) in 2 others, both non-carriers of the ApoE4 allele.• ApoE4 carriers were significantly older than the non-carriers (p=0.006) and possessed more years of education (p=0.092).• Nadir CD4+ T-cell count (the lowest ever reached) was significantly higher in ApoE4 carriers (p=0.031) than in non-carriers after adjusting for age. Current CD4 count did not differ between groups (p=0.94). (CD4 data was missing for one ApoE4-negative subject.)• NART FSIQ (National Adult Reading Test Full-Scale Intelligence Quotient) did not differ between ApoE4 carriers and non-carriers (p=0.99)• Dementia status had not been assigned to many in our cohort; however, a quick review of z-transformed neuropsychological test scores indicates that very few, if any, would be classed as demented.

Reduced Region-Specific Corpus Callosum Volumes Correlate with Low Nadir CD4 and Reduced Region-Specific Corpus Callosum Volumes Correlate with Low Nadir CD4 and Decreased Cognition in HIV-Infected Carriers of the ApoE4 AlleleDecreased Cognition in HIV-Infected Carriers of the ApoE4 Allele

Kalpana J Kallianpur*1, Aaron McMurtray1, Victor Valcour1,2,Bruce Shiramizu1 and Cecilia Shikuma1 1 Hawaii AIDS Clinical Research Program, University of Hawaii; 2 University of California–San Francisco

ApoE4 and Cognitive Impairment

RESULTS

In HIV infection, reduced regional and total corpus callosum volumes correlate with low nadir CD4 in carriers (but not in non-carriers) of the ApoE4 allele. Callosal volumes and ApoE4 presence are also associated with performance on certain neurocognitive tests. Should these data be confirmed, research to determine if prompt initiation of HAART can modify the above-described relationships may be advisable. Further investigation using large ApoE4-positive and -negative groups matched for CD4 nadir is indicated.

Apolipoprotein E is essential to the metabolism and transport of serum lipids, and mediates cholesterol metabolism in the brain. The E4 isoform (ApoE4) has been linked to impaired cognitive function in the non-demented elderly¹, to vascular dementia², and to increased vulnerability to late-onset Alzheimer’s Disease (AD)³. Inheritance of a single ApoE4 allele nearly doubles an individual’s lifetime risk of developing AD4. Diffusion tensor imaging has revealed compromised white matter integrity in the posterior corpus callosum of healthy non-demented ApoE4 carriers5; and even in healthy, cognitively normal older individuals, presence of the allele has been shown to affect implicit learning6.

Region-specific atrophy of the corpus callosum has been implicated in AD, mild neurocognitive impairment, and cognitive complaints of older adults with intact neuropsychological functioning7. When impairment is slight the posterior callosal region may be the most affected, with atrophy progressing to other regions as dementia grows more severe. Callosal atrophy has also been linked to impaired cognitive and motor function in healthy elderly subjects with age-related white matter changes8.

As previously reported by our group, HIV-infected patients who are over 50 years of age and heterozygous for ApoE4 have a higher risk of developing HIV-associated dementia than do non-carriers of the allele9. Nadir CD4 count has been proposed as a predictor of current cognitive and neurological status10. A recent comparison of four nadir CD4 cutoff points (200 to 350 cell/ml) found a trend toward a higher prevalence of impairment as CD4 nadir decreased, along with significantly greater deficits in attention, working memory and executive function11. Relative to HIV-seronegative controls, non-demented AIDS patients show a trend toward lower total callosal volume as well as significantly reduced thickness throughout the region, with the thinning in anterior sections correlated to CD4 T-cell decline12. Diffusion abnormalities detected in the genu and splenium have been associated with cognitive and motor speed effects of HIV infection13,14. The present study investigates the relationship among neurocognitive test performance, corpus callosum volume, nadir CD4 and presence of an ApoE4 allele.

Kalpana J. Kallianpur, Ph.D.Hawaii AIDS Clinical Research ProgramLeahi Hospital3675 Kilauea Ave. Young Bldg 5th FloorHonolulu, HI 96816Email: kalpana@hawaii.eduPh: (808) 232-8128Fax: (808) 735-7047

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Subjects underwent standard neuropsychological testing that included the Rey Complex Figure Test (Copy, Immediate Recall and Delayed Recall tasks). ANOVA of test scores by ApoE4 category suggests that non-verbal memory and visuospatial ability are significantly diminished in HIV-infected patients carrying the ApoE4 allele. Presence of the ApoE4 allele was also associated with significantly poorer performance on the Rey Auditory Verbal Learning Test (List B), Grooved Pegboard Tests for dominant and non-dominant hand, Wechsler Adult Intelligence Scale (WAIS) Digit-Symbol Modalities (DSM), WAIS Letter-Number Sequencing, and Trail-Making Test B. These results are based on raw rather than on z-transformed test scores and must not be taken as conclusive.

• In a cross-sectional study, 17 older (age > 50 years) HIV-seropositive patients underwent MRI on a 3.0 Tesla Philips scanner. Pulse sequence parameters for T1-weighted images were TR=6.7 ms, TE=3.1 ms, and flip angle of 8°, with an image slice thickness of 1.2 mm and in-plane resolution of 1.0 mm2.• FreeSurfer software was utilized to process the T1-weighted images. The workflow included estimation of intracranial volume (ICV) and white matter hyperintensity (WMH) volume.• StatView (v.5, SAS Institute, Inc.) was used to execute T-tests, analysis of covariance (ANCOVA), and regression for statistical analysis of callosal volumetric data and their possible link to nadir CD4 levels.• The corpus callosum was segmented into five labels: posterior, mid-posterior, central, mid-anterior and anterior. Regional and total callosal volumes were divided by ICV to adjust for head or overall brain size. Statistical analyses were done on normalized volumes.• Neuropsychological testing of patients was performed within 30 days of MRI. • Determination of ApoE genotype involved9 :

o Restriction enzyme of the 227 base pair PCR product of each specimen o Digestions with /HhaI, HaeII, and AflIII; /followed by resolution by gel electrophoresis

• Statistical significance is defined by p < 0.05, and trends by 0.05 ≤ p < 0.1. Due to small sample size and the exploratory nature of this work, p-values were not corrected for multiple comparisons.

Total and regional corpus callosum volumes were normalized to ICV, which did not differ between ApoE4 carriers and non-carriers (p=0.64). ANOVA by ApoE4 group status showed no significant differences in normalized callosal volumes between carriers and non-carriers of the ApoE4 allele (e.g., total corpus callosum: p=0.86). When age, current CD4 count, education and WMH volume were each included separately as covariates, the last two variables had significant main effects but ApoE4 group differences in callosal volumes remained non-significant.

Characteristics of Cohort

BACKGROUND

Task: Immediate Recall

0

10

20

Test

Sco

re

p = 0.023

A connection between reduced corpus callosum volume and low nadir CD4 count was observed only in individuals possessing an ApoE4 allele; CD4 nadir correlated positively with genu (p=0.40), splenium (p=0.057), mid-posterior (p=0.048) and total (p=0.57) callosal volumes. These volumes were associated with visuospatial function and non-verbal memory test scores. No relation was found between callosal volume and nadir CD4 in ApoE4-negative patients. HIV-infected patients who carry an ApoE4 allele may therefore be at increased risk of neurocognitive impairment stemming from corpus callosum atrophy.

Prior research has linked both low nadir CD4 and the ApoE4 allele with diminished cognition. The significantly higher CD4 nadir levels in our ApoE4-positive group compared to non-carriers (Figure 2) probably obscured the relationship between ApoE4 presence and nadir CD4 in this study. For example, we found regional callosal volumes to be associated with performance on only three neuropsychological tests (Table 3) out of the nine that showed significant differences between the two ApoE4 groups. Furthermore, although test scores showed main effects of ApoE4 and callosal volumes, interactions between the volumes and ApoE4 presence were not significant. These results appear likely to change if ApoE4-positive and -negative study participants are well-matched for nadir CD4, particularly because regional callosal volumes did show ApoE4-nadir CD4 interaction effects (Figure 3).

Small sample size limited our ability to adjust for variables such as white matter lesion volume and duration of HIV infection. A larger study population, matched for CD4 nadir count, should be recruited for future work. Improved analytical techniques (e.g., use of normalized neuropsychological test scores, and more accurate estimates of ICV and WHM volume) and other imaging modalities (e.g., diffusion tensor imaging) will help to clarify associations among cognitive deficits, low nadir CD4, corpus callosum atrophy and presence of an ApoE4 allele.

Corpus Callosum Volume Dependence on CD4 Nadir: Possible ApoE4 Effect

ApoE4 carriers: p=0.057, R=0.69 Non-carriers: NS

(p=0.56)

ApoE4 and Corpus Callosum Volume

Cognitive domain

TestApoE4 ( p-value)

Non-verbal memory

Rey IR 0.023

Rey DR 0.028

Visuospatial function

Rey Copy

0.012

Cognitive domain

Test p-value

ApoE4

Regional callosal volume

Non-verbal

memory

Rey IR and DR (mean)

0.022

0.055 (anterior)

0.011

0.088 (central)

0.012

0.089 (posterior)

0.012

0.058 (total)

Visuospatial ability

Rey Copy

0.003

0.025 (posterior)

0.004

0 .048 (mid-posterior)

0.005

0.075 (central)

0.006

0.061 (total)

Task: Delayed Recall

0

10

20Te

st S

core Non-carriers

ApoE4 carriers

Performance on Rey Complex Figure Test

Task: Copy

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30

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Right: results of ANCOVA for neuropsychological test scores that showed effects of corpus callosum volume. Rey Copy and the mean of Rey IR and DR test scores were outcomes, ApoE4 group status the factor, and regional callosal volumes (separately) the independent variables. We did not control for age or NART FSIQ, as they were not correlated with these test scores (and were verified to have no significant main effects). Education was not included as a covariate because it correlated with callosal volumes and because of small sample size.

(For RAVLT (List B), Grooved Pegboard Test, Trail-Making B, and WAIS DSM and LN-Sequencing test scores, similar ANCOVA was performed. Age correlated with test performance; after controlling for age, no regional corpus callosum volume had a significant or trend-level effect on these test scores.)

Figure 1. Mean of raw test scores. Error bars represent standard error of the mean.

p = 0.028

p = 0.012

ANCOVA of callosal sectional volumes by ApoE4 category, with CD4 nadir as an independent variable, indicated a trend main effect of CD4 nadir (p=0.061) but not of ApoE4 group status on the volume of the mid-posterior region. Effects on callosal volumes of CD4 nadir interaction with ApoE4 allele presence were not significant: interaction p-values for anterior (genu), posterior (splenium), and total corpus callosum volume were 0.11, 0.10 and 0.16, respectively. The ANCOVA may be of questionable validity because nadir CD4 levels are associated with ApoE4 category in our study population (Figure 2).

CD4 nadir (cells/mm3)

Vo

lum

e (%

IC

V)

Posterior corpus callosum (splenium)

.03

.035

.04

.045

.05

.055

.06

.065

.07

.075

0 50 100 150 200 250 300 350 400 450 500

.02

.03

.04

.05

.06

.07

.08

0 50 100 150 200 250 300 350 400 450 500

CD4 nadir (cells/mm3)

Anterior corpus callosum (genu)

Table 2. ANOVA: effect of ApoE4 allele presence on selected raw

test scores.

ApoE4 carriers: p=0.040, R=0.73 Non-carriers: NS

(p=0.78)

Table 1. Selected demographic and clinical parameters.

Table 3. ANCOVA of raw neuropsychological test scores by ApoE4 category. Independent variables: callosal volumes.

Total corpus callosum

ApoE4 carriers: p=0.048, R=0.712 Non-carriers: NS

(p=0.46)

(Combined: p=0.10, R=0.42)

Mid-posterior corpus callosum

ApoE4 carriers: p=0.057, R=0.692 Non-carriers: NS

(p=0.77)

ApoE4 carriers

Non-carriers

Figure 3. Lines represent linear regression of corpus callosum volumes on CD4 nadir.

Figure 3 shows the direct relationship of CD4 nadir to genu, splenium, mid-posterior and total corpus callosum volumes for subjects in both ApoE4 groups:

Overall (N = 17)

ApoE4-positive (N=8)

ApoE4-negative (N=9)

Gender (%) Male 17 (100) 8 (100) 9 (100)

Age (years) Mean (S.D.) 55.8 (5.2) 63.1 (6.1) 53.9 (5.6)

Education (years) Mean (S.D.) 15.1 (2.4) 16.5 (2.3) 14.7 (1.9)

WMH (%) Present 6 (35.3) 4 (50) 2 (22.2)

Race/Ethnicity (%) Caucasian 13 (76.5) 7 (87.5) 6 (67)

Asian/Pacific Islander 2 (12) 0 (0) 2 (22)

Hispanic 1 (6) 0 (0) 1 (11)

Native American/Native Alaskan 1 (6) 1 (12.5) 0 (0)

Current CD4 (cells/mm3) Mean (S.D.) 414.4 (148.7) 388.8 (183.5) 396.3 (175.4)

Nadir CD4 (cells/mm3) Mean (S.D.) 104.9 (103.6) 206.5 (136.3) 112.1 (106.4)

Regions of the corpus callosum.

Anterior

Posterior

Mid-anterior.

Mid-posterior

Central

Nadir CD4

050

100150200250

Non-carriers ApoE4 carriers

Q. Do ApoE4 group status and regional corpus callosum volumes have a combined effect on cognition?

Q. Do ApoE4 group status and nadir CD4 count have a combined effect on regional corpus callosum volumes?

As expected, HIV-infected patients carrying an ApoE4 allele show neurocognitive deficits when compared with non-carriers.

A. Regional callosal volumes (particularly posterior) affect test scores of non-verbal memory and visuospatial function, but presence of an ApoE4 allele is more significant. Interactions between ApoE4 group status and callosal volumes were not significant here. Tests other than Rey Complex Figure Test (IR, DR and Copy) showed no significant effects of corpus callosum volume.

A. Among ApoE4 carriers only, anterior and mid-posterior callosal volumes correlate significantly with nadir CD4, and posterior and total corpus callosum volumes show a trend toward such an association. Small sample size and unbalanced nadir CD4 count between the two ApoE4 groups are likely to be masking the true relationship among callosal volume, CD4 nadir and presence of the ApoE4 allele.

Figure 2. Mean nadir CD4 count is 84% higher in ApoE4-positive group than for ApoE4-negative patients. After controlling for age, the difference is significant (p=0.031).

p = 0.031

Summary

1. P. Bretsky, J. M. Guralnik, L. Launer, M. Albert, and T. E. Seeman. The role of APOE-4 in longitudinal cognitive decline: MacArthur Studies of Successful Aging. Neurology, 2003 Apr 8;60(7):1077-81.2. Frisoni GB, Manfredi M, Geroldi C, Binetti G, Zanetti O, Bianchetti A, Trabucchi M. The prevalence of apoE-epsilon4 in Alzheimer's disease is age dependent. J Neurol Neurosurg Psychiatry, 1998 Jul;65(1):103-6.3 . Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, Roses AD, Haines JL, Pericak-Vance MA (1993). Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families. Science 261 (5123): 921–3.4. Mayeux R, Saunders AM, Shea S, Mirra S, Evans D, Roses AD, Hyman BT, Crain B, Tang MX, Phelps CH. Utility of the apolipoprotein E genotype in the diagnosis of Alzheimer's disease. Alzheimer's Disease Centers Consortium on Apolipoprotein E and Alzheimer's Disease. N Engl J Med. 1998 Feb

19;338(8):506-115. Persson J, Lind J, Larsson A, Ingvar M, Cruts M, Van Broeckhoven C, Adolfsson R, Nilsson LG, Nyberg L. Altered brain white matter integrity in healthy carriers of the APOE epsilon4 allele: a risk for AD? Neurology. 2006 Apr 11;66(7):1029-336. S. Negash, L. Petersen, Y. Geda, D. Knopman, B. Boeve, G. Smith, R. Ivnik, D. Howard, J. Howard, Jr., R. Petersen. Effects of ApoE genotype and mild cognitive impairment on implicit learning. Neurobiology of Aging, 2007, Volume 28, Issue 6, Pages 885-8937. Wang P, Saykin A, Flashman L, Wishart H, Rabin L, Santulli R, McHugh T, MacDonald J, Mamourian A. Regionally specific atrophy of the corpus callosum in AD, MCI and cognitive complaints. Neurobiol Aging. 2006 Nov;27(11):1613-7.8. C. Ryberg, E. Rostrup, M. B. Stegmann, F. Barkhof, P. Scheltens, E. C. W. van Straaten, F. Fazekas, R. Schmidt, J. M. Ferro, H. Baezner, T. Erkinjuntti, H. Jokinen, L. Wahlund, J. O’Brien, A. M. Basile, L. Pantoni, D. Inzitari, G. Waldemar. Clinical significance of corpus callosum atrophy in a mixed elderly

population, Neurobiol Aging. 2007 Jun;28(6):955-63. Epub 2006 Jun 23.9. V. Valcour, C. Shikuma, B. Shiramizu, M. Watters, P. Poff, O.A. Selnes, J. Grove, Y. Liu, K.-B. Abdul-Majid, S. Gartner, N. Sacktor. Age, apolipoprotein E4, and the risk of HIV dementia: the Hawaii Aging with HIV Cohort. Journal of Neuroimmunology - December 2004 (Vol. 157, Issue 1: 197-202)10. Valcour V, Yee P, Williams AE, Shiramizu B, et al.: Lowest ever CD4 lymphocyte count (CD4 nadir) as a predictor of current cognitive and neurological status in human immunodeficiency virus type 1 infection -The Hawaii Aging with HIV Cohort. J Neurovirol 2006;12(5):387-391.11. J.A. Muñoz-Moreno, C.R. Fumaz, M.J. Ferrer, A. Prats, E. Negredo, M. Garolera, N. Pérez-Álvarez, J. Moltó, G. Gómez, B. Clotet. (2008) Nadir CD4 Cell Count Predicts Neurocognitive Impairment in HIV-Infected Patients. AIDS Research and Human Retroviruses 24:10, 1301-130712. Thompson, P.M., Dutton, R.A., Hayashi, K.M., Lu, A., Lee, S.E., Lee, J.Y., Toga, A.W., Lopez, O.L., Aizenstein, H.J., Becker, J.T. (2006). 3D Mapping of Ventricular and Corpus Callosum Abnormalities in HIV/AIDS, NeuroImage, Jan. 200613. David Tate, Robert Paul, Kathryn Coop, Timothy Flanigan, Song Zhang, David H. Laidlaw, and Karen Tashima. Altered Diffusion Scalar Metrics in the Midsagittal Corpus Callosum are Associated with Cognition Among HIV Patients. In Proceedings of the 14th Conference on Retroviruses and Opportunistic

Infections, Los Angeles, CA, 2006.14. Wu Y, Storey P, Cohen BA, Epstein LG, Edelman RR, Ragin AB. Diffusion Alterations in Corpus Callosum of Patients with HIV. AJNR Am J Neuroradiol. 2006 Mar;27(3):656-60.

CD4 nadir (cells/mm3) CD4 nadir (cells/mm3)

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.015

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.14

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Acknowledgement:  This project received support from P20 RR11091 NIH/NCRR RCMI Clinical Research Infrastructure Initiative Subproject #2007-07: Apolipoprotein E4 Status and Cortical Metabolism in HIV Infection; the American Academy of Neurology Foundation Clinical Research Training Fellowship Award (CRTF); and U19MH081835: Macrophage Targeted Therapy for HAD and HIV Disease.